Comparison of egg and high yielding MDCK cell-derived live attenuated influenza virus for commercial production of trivalent influenza vaccine: in vitro cell susceptibility and influenza virus replication kinetics in permissive and semi-permissive cells

Abstract

Currently MedImmune manufactures cold-adapted (ca) live, attenuated influenza vaccine (LAIV) from specific-pathogen free (SPF) chicken eggs. Difficulties in production scale-up and potential exposure of chicken flocks to avian influenza viruses especially in the event of a pandemic influenza outbreak have prompted evaluation and development of alternative non-egg based influenza vaccine manufacturing technologies. As part of MedImmune's effort to develop the live attenuated influenza vaccine (LAIV) using cell culture production technologies we have investigated the use of high yielding, cloned MDCK cells as a substrate for vaccine production by assessing host range and virus replication of influenza virus produced from both SPF egg and MDCK cell production technologies. In addition to cloned MDCK cells the indicator cell lines used to evaluate the impact of producing LAIV in cells on host range and replication included two human cell lines: human lung carcinoma (A549) cells and human muco-epidermoid bronchiolar carcinoma (NCI H292) cells. The influenza viruses used to infect the indicators cell lines represented both the egg and cell culture manufacturing processes and included virus strains that composed the 2006-2007 influenza seasonal trivalent vaccine (A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/05 (H3N2) and B/Malaysia/2506/04). Results from this study demonstrate remarkable similarity between influenza viruses representing the current commercial egg produced and developmental MDCK cell produced vaccine production platforms. MedImmune's high yielding cloned MDCK cells used for the cell culture based vaccine production were highly permissive to both egg and cell produced ca attenuated influenza viruses. Both the A549 and NCI H292 cells regardless of production system were less permissive to influenza A and B viruses than the MDCK cells. Irrespective of the indicator cell line used the replication properties were similar between egg and the cell produced influenza viruses. Based on these study results we conclude that the MDCK cell produced and egg produced vaccine strains are highly comparable.

Fig. 1

Comparison of replication kinetics of egg and cell produced influenza A and influenza B virus infected MDCK cells. Kinetics of CP and EP cold-adapted A/New Caledonia/20/99, A/Wisconsin/67/05 and B/Malaysia/2506/04 influenza virus replication in MDCK cells measured by FFA (each data point represents average of triplicate samples tested).

Fig. 2

Comparison of viral RNA copy number of egg and cell produced influenza A and B virus infected MDCK, A549 and NCI H292 cells. Kinetics of CP and EP cold-adapted A/New Caledonia/20/99, A/Wisconsin/67/05 and B/Malaysia/2506/04 influenza virus M segment RNA in MDCK, A549 and NCI H292 cells measured by qRT-PCR (each data point represents average of triplicate samples tested). The maximum SD for the triplicates for MDCK, A549 and NCI H292 cells was found to be 0.23, 0.48 and 0.43, respectively.

Fig. 3

Comparison of influenza A and B viral RNA accumulation in infected MDCK cells. Kinetics of CP and EP cold-adapted A/New Caledonia/20/99 (A/NC), A/Wisconsin/67/05 (A/Wis) and B/Malaysia/2506/04 (B/Mal) influenza virus M, NP and PB1 segment RNA in MDCK cell lysates by qRT-PCR (each data point represents average of triplicate samples tested). The maximum SD for the triplicates for A/NC, A/Wis and B/Mal was found to be 0.13, 0.22 and 0.09, respectively.

Fig. 4

Comparison of influenza A and B viral RNA accumulation in infected A549 cells. Kinetics of CP and EP cold-adapted A/New Caledonia/20/99 (A/NC), A/Wisconsin/67/05 (A/Wis) and B/Malaysia/2506/04 (B/Mal) influenza virus M, NP and PB1 segment RNA in A549 cell lysates by qRT-PCR (each data point represents average of triplicate samples tested). The maximum SD for the triplicates for A/NC, A/Wis and B/Mal was found to be 0.17, 0.10 and 0.09, respectively.

Fig. 5

Comparison of influenza A and B viral RNA accumulation in infected NCI H292 cells. Kinetics of CP and EP cold-adapted A/New Caledonia/20/99 (A/NC), A/Wisconsin/67/05 (A/Wis) and B/Malaysia/2506/04 (B/Mal) influenza virus M, NP and PB1 segment RNA in NCI H292 cell lysates by qRT-PCR (each data point represents average of triplicate samples tested). The maximum SD for the triplicates for A/NC, A/Wis and B/Mal was found to be 0.17, 0.13 and 0.08, respectively.

Fig. 6

Comparison of progression of infection observed using immunofluorescence in egg and cell produced influenza A and B virus infected MDCK cells (MOI 0.1). Kinetics of CP and EP cold-adapted A/New Caledonia/20/99 influenza virus replication measured by Immunofluorescence assay on MDCK 9B91E4 (magnification 50×).

Fig. 7

Viral protein expression in MDCK cells infected with cell and egg produced virus. A/New Caledonia/20/99. Kinetics of viral protein expression in CP and EP cold-adapted A/New Caledonia/20/99 MDCK cells detected using sheep antibodies show the major HA proteins including HA0, HA1 and HA2 proteins.